Combination treponemal and non-treponemal syphilis test

ABSTRACT

Provided herein are multiplex assays for detecting antibodies indicative of presence and stage of syphilis infection in an individual. Individuals infected with syphilis produce antibodies directed to syphilis components and the lipid cellular debris associated with the infection. The present disclosure represents the first combination of these diverse antibody targets in a single assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 15/701,272, filed on Sep. 11, 2017, which is acontinuation application of International Application No.PCT/US2016/021379, filed on Mar. 8, 2016, which claims benefit ofpriority to U.S. Provisional Patent Application No. 62/131,062, filedMar. 10, 2015 and U.S. Provisional Patent Application No. 62/181,685,filed Jun. 18, 2015, each of which is incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Syphilis is a sexually transmitted disease (STD) caused by thespirochete bacterium Treponema pallidum . Serological tests for syphiliscan be classified into two groups, nontreponemal and treponemal tests.Nontreponemal tests detect nontreponemal antibodies (reagin) againstlipoidal material released from damaged host cells. The most widely usednontreponemal tests are Venereal Diseases Research Laboratory (VDRL) andrapid plasma reagin (RPR) tests, which are tests (usually flocculationtests) employing a cardiolipin-lecithin-cholesterol-based antigen.Nontreponemal tests have the advantage of being widely available,inexpensive and convenient to perform. Results for these tests usuallyrevert to negative after successful treatment, so they can be used tomonitor response to therapy. These tests typically require subjectiveinterpretation however, so they are difficult to interpret, particularlyfor weakly reactive sera. The nontreponemal tests also have limitedpotential for high-volume testing and automation. In contrast, thetreponemal tests detect antibodies directed against native orrecombinant surface antigens of T pallidum such as 15 kDa, 17 kDa, and47 kDa proteins. Treponemal tests include T pallidum passive particleagglutination (TP-PA) assay, the fluorescent treponemal antibodyabsorption (FTA-ABS) test, and most enzyme immunoassays (EIA) tests.Compared to nontreponemal tests, treponemal tests exhibit higherspecificity and sensitivity. They cannot, however, be used to monitorresponse to therapy because treponemal antibodies remain for years afterinfection.

Syphilis is diagnosed using either traditional algorithm (patientspecimen screened by nontreponemal test followed by tremonemal testconfirmation) or reverse algorithm (patient specimen screened bytreponemal test followed by nontreponemal confirmation). Essentially,both algorithms follow a positive initial screen with a confirmationtest.

BRIEF SUMMARY OF THE INVENTION

Provided herein are kits to detect nontreponemal and treponemalantibodies in a sample from an individual suspected of having a syphilisinfection. In some embodiments, the kit includes (i) a solid support(e.g., microparticles, beads, or surface such as a chip, microtiterplate, membrane, or glass) coated with a lipoidal antigen (e.g.,cardiolipin, lecithin, cholesterol, or a mixture thereof); and (ii) asolid support (e.g., microparticles, beads, or surface such as a chip,microtiter plate, membrane, or glass) coated with Treponema pallidumantigen. In some embodiments, (i) and (ii) are packaged in the samecompartment (e.g., in the same mixture in a vessel or tube, or coated onthe same surface, e.g., of an ELISA well). In some embodiments, (i) and(ii) are packaged in separate compartments. In some embodiments, thebeads are in storage buffer comprising pH buffer (e.g., Trizma, glycine,phosphate) and salt (e.g., NaCl). In some embodiments, the storagebuffer further includes an ionic or zwitterionic detergent. In someembodiments, the kit further comprises an assay buffer and/or sampledilution buffer. In some embodiments, the storage buffer, the assaybuffer, and/or sample dilution buffer lacks non-ionic detergent.

In some embodiments, the lipoidal antigen is cardiolipin, lecithin orcholesterol. In some embodiments, the Treponema pallidum antigen is r15,r17, r47, a mixture thereof, or a fusion of r17 and r47.

In some embodiments, the kit further includes a labeledanti-immunoglobulin (Ig) antibody, e.g., an anti-human Ig antibody. Insome embodiments, the anti-Ig antibody is specific for IgG (e.g., humanIgG) and/or IgM (e.g., anti-human IgM). In some embodiments, thelabeled, anti-human immunoglobulin antibody is an antibody conjugatethat binds human IgG and human IgM. In some embodiments, the kitcomprises a first labeled, anti-human Ig antibody specific for IgG and asecond labeled, anti-human Ig antibody specific for IgM. The anti-humanIgG antibody and anti-human IgM antibody can have the same or differentlabels.

In some embodiments, the kit further includes a wash solution or washstock solution (e.g., concentrate or dehydrated). In some embodiments,the wash solution or wash stock solution includes PBS, and optionally anionic, non-ionic, or zwitterionic detergent. In some embodiments, thewash solution has non-ionic detergent. In some embodiments, the washsolution lacks non-ionic detergent.

In some embodiments, the lipoidal antigen is coupled (attached) to thesolid support of (i) via an ionic interaction. In some embodiments, thesolid support of (i) is coated with polyethylenimine (PEI). In someembodiments the Treponema pallidum antigen is coupled to the solidsupport of (ii) via a covalent interaction. In some embodiments, thesolid supports (e.g., microparticles or beads) of (i) and (ii) aredifferently labeled, of different sizes, or of different weights.

In some embodiments, the kit further comprises at least one controlagent. In some embodiments, the control agent is an internal controlagent, e.g., to be used in the same reaction tube or vessel with theassay reagents and sample. In such case, the internal control isdistinguishable from other solid supports, e.g., with a distinct labelor weight, as described in more detail herein. In some embodiments, thecontrol reagent is a solid support, wherein the solid support isconjugated to an agent that binds specimen specific analyte (e.g., aspecimen verification control, e.g., a serum verification bead). Thus insome embodiments, the kit further comprises (iii) beads coated with asample quality control reagent (e.g., a serum verification bead (SVB)coated with a reagent that reacts with common serum element, such as anantibody specific for a coagulation factor (e.g., FXIII), globulin, oralbumin)). In some embodiments, the kit further comprises a controlcomprising a solid support lacking reagent as a blank, e.g., (iv) areagent blank bead (RBB). In some embodiments, the kit further comprisesa control to determine the quality of signal, e.g., a solid supportlabeled with a known label or combination of labels. For example, insome embodiments, the kit includes (v) a signal quality control bead(e.g., an internal standard bead (ISB) with intrinsic fluorescence). Insome embodiments, the kit only includes one of (iii), (iv), or (v). Insome embodiments, the kit includes (iii), (iv), and (v). In someembodiments, the kit includes any combination of two of the controls(e.g., (iii) and (iv), (iii) and (v), or (iv) and (v)).

Further provided are methods of detecting the presence of syphilisantibodies in a sample, e.g., a biological sample from an individual,e.g., a human. In some embodiments, the method comprises (a) contactingthe sample with (i) solid support (e.g., microparticles, beads, orsurface) coated with lipoidal antigen (e.g.,cardiolipin-lecitin-cholesterol antigen) and (ii) solid support (e.g.,microparticles, beads, or surface) coated with Treponema pallidumantigen in the same compartment, thereby forming a sample-support (e.g.,sample-bead) mixture; (b) washing the sample-support mixture to removeunbound sample; (c) contacting the sample-support mixture with alabeled, anti-immunoglobulin (Ig) antibody (e.g., anti-human Igantibody); and (d) detecting the presence of syphilis antibodies bydetecting the label on the anti-Ig antibody. In some embodiments, step(a) and/or step (c) are carried out in a buffer that does not includenon-ionic detergent.

In some embodiments, the method further comprises washing to removeunbound anti-Ig antibody between steps (c) and (d). In some embodiments,the washing is carried out with non-ionic detergent. In someembodiments, the washing is carried out in the absence of non-ionicdetergent. In some embodiments, the washing is carried out withphosphate buffered saline (PBS), optionally with ionic, non-ionic, orzwitterionic detergent. In some embodiments, non-ionic detergent is usedin wash buffers in the presently described assays, but at a lowerconcentration than in standard assays having hydrophilic (non-lipid)components. In some embodiments, washing with non-ionic detergent iscarried out with reduced duration compared to standard assays havinghydrophilic (non-lipid) components (e.g., 2-20, 1-10, or 2-8 seconds,compared to 10-60, or 30-90 seconds). In some embodiments, fewer washesare carried out with non-ionic detergent compared to standard assayshaving hydrophilic (non-lipid) components (e.g., 2-4, 2, 3, or 4 timesover duration of assay, compared to 4, 8, or 5-10 times).

In some embodiments, the labeled anti-Ig antibody is anti-human Ig. Insome embodiments, the labeled anti-human Ig antibody is specific for IgGand/or IgM. In some embodiments, the labeled, anti-human immunoglobulinantibody is an antibody conjugate that binds human IgG and human IgM. Insome embodiments, step (c) comprises contacting the sample-supportmixture with a first labeled, anti-human Ig antibody specific for IgGand a second labeled, anti-human Ig antibody specific for IgM. The firstand second labeled antibodies can be labeled with the same or withdifferent labels.

In some embodiments, the method further comprises prescribing a courseof treatment if syphilis antibodies are detected in step (d). In someembodiments, course of treatment includes treatment with an antibiotic(e.g., penicillin, tetracycline, doxycycline, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the schematic illustration of a combo test for thedetection of both treponemal and nontreponemal antibodies on anautomated platform (e.g., BioPlex™ 2200).

FIG. 2 depicts bead surface functionalization to allow the beads to becoated with lipid nontreponemal antigen.

DETAILED DESCRIPTION OF THE INVENTION A. INTRODUCTION

Described herein is a novel combination test for simultaneous detectionof treponemal and nontreponemal antibodies in patient samples. Theassays can be performed efficiently on an automated platform. Thecombination test comprises a treponemal test (e.g., beads labeled with afirst label and coated with T pallidum recombinant r17 and r47 fusionprotein), and a nontreponemal test (e.g., beads labeled with a secondlabel and an antigen mixture of cardiolipin-lecithin-cholesterol). Thecombination test can also comprise labeled anti-human IgG and labeledanti-human IgM reporters (e.g., anti-hIgG-PE and anti-hIgM-PE) toquantify the treponemal and nontreponemal antibodies in the specimen.

The combination of (i) beads covalently linked to antigen (treponemal)with (ii) beads non-covalently coated with a lipid antigen(nontreponemal) in a single assay or kit is not straightforward becausedifferent conditions are generally used for storage and washing of thetwo types of beads. Using the presently disclosed conjugation techniquesand buffer conditions, however, the combined reagents are stable for atleast 12-24 months. In addition, the assays are sensitive and accuratein spite of lack of non-ionic detergent in the storage solution (inwhich the solid support or beads are stored), sample dilution buffer,and assay solution (in which sample is incubated with the beads).Typically, non-ionic detergents are used in various solutions to reducebackground signal, but as shown herein, assays combining bothamine-functionalized beads coated with hydrophobic nontreponemal antigenand beads coated with hydrophilic treponemal antigen detected antibodieswith high accuracy in Syphilis patients at various stages of infectionand treatment. In some embodiments, non-ionic detergent is used in washbuffers in the presently described assays, but at a lower concentrationthan in standard assays having hydrophilic (non-lipid) components. Insome embodiments, washing with non-ionic detergent is carried out withreduced duration or frequency compared to standard assays havinghydrophilic (non-lipid) components.

Combining the two types of beads with different affinities andcomplexity involved process modification, such as omitting detergentsfrom the sample diluent. The two types of beads would typically bestored and used in distinct conditions; no one has combined the twoformats i.e., hydrophilic and hydrophobic micro environments in oneassay.

Further, the results described herein identify reagin-specific IgM andIgG antibodies using IgM-PE and IgG-PE conjugates. This approach hashelped detect the presence of IgM antibodies among reagin positivepatients. Indeed, we determined that the majority of anti-reaginantibodies are of the IgM isotype. Current assays reportreactive/non-reactive results without identifying the isotype of theantibodies. IgM isotype antibodies are indicative of early stage acuteinfection, and IgG isotype antibodies become prevalent later. Loss ordecrease of RPR specific IgM antibodies can allow a physician to monitorand prescribe drug treatment appropriately (e.g., prescribing a higheror more frequent dose of antibiotics for more advanced infections). Insome embodiments, stage can be determined by detecting anti-IgM-PE oranti-IgG-PE alone, or by using different labels on the anti-Igantibodies.

B. DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989).

Multiplex assays are analyses that simultaneously measure the levels ofmore than one analyte in a single sample. Multiplex assay methods andreagents are described, e.g., in U.S. Pat. No. 6,773,578 andWO2008148883.

The term “solid support” is used herein to denote a solid inert surfaceor body to which an agent, such as an antibody or an antigen can beimmobilized. Non-limiting examples include plastic, nitrocellulose,membranes, chips, magnetic or non-magnetic beads, and particles. Theterm “immobilized” as used herein denotes a molecular-based couplingthat is not significantly de-coupled under the conditions imposed duringthe steps of the assays described herein. Such immobilization can beachieved through a covalent bond, an ionic bond, an affinity-type bond,or any other chemical bond.

The term “particle” is used herein to refer to a solid or semisolidbody, often with linear dimensions on the micron scale (i.e., less than100 microns), of any shape or surface texture. Except as noted, the termis used interchangeably with “microparticle,” which refers to a micronscale particle, and “bead,” which refers to particles that are sphericalor near-spherical in shape, often polymeric in composition.

The terms “receptacle,” “vessel,” “tube,” “well,” “compartment,” etc.refer to a container that can hold reagents or an assay. If thereceptacle is in a kit and holds reagents, it will typically be closedor sealed. If the receptacle is being used for an assay, it willtypically be open or accessible during steps of the assay.

The terms “sample” and “biological sample” encompass a variety of sampletypes obtained from an organism. The term encompasses bodily fluids suchas saliva, sputum, blood, blood components, serum, plasma, urine andother liquid samples of biological origin, solid tissue biopsy, tissuecultures, or supernatant taken from cultured patient cells. In thecontext of the present disclosure, the biological sample is typically abodily fluid with detectable amounts of antibodies, e.g., sputum, mucus,mucosal tissue biopsy, or scrape. The biological sample can be processedprior to assay, e.g., to remove cells or cellular debris. The termencompasses samples that have been manipulated after their procurement,such as by treatment with reagents, solubilization, sedimentation, orenrichment for certain components.

The term “antibody” as used herein refers to a polypeptide encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin light chains are classified as either kappa or lambda.Immunoglobulin heavy chains are classified as gamma, mu, alpha, delta,or epsilon, which in turn define the immunoglobulin classes, IgG, IgM,IgA, IgD and IgE, respectively. In humans, there are four subclasses ofIgG, denoted IgG1, IgG2, IgG3, and IgG4.

An example of a structural unit of immunoglobulin G (IgG antibody) is atetramer. Each such tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(VL) and “variable heavy chain” (VH) refer to these light and heavychains, respectively.

Antibodies exist as intact immunoglobulins or as well-characterizedfragments produced by digestion of intact immunoglobulins with variouspeptidases. Thus, for example, pepsin digests an antibody near thedisulfide linkages in the hinge region to produce F(ab′)2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab′)2 dimer can be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab′)2dimer into two Fab′ monomers. The Fab′ monomer is essentially an Fabwith part of the hinge region (see, Paul (Ed.), Fundamental Immunology,Third Edition, Raven Press, N.Y. (1993)). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such fragments may be synthesized denovo either chemically or by utilizing recombinant DNA methodology.Thus, the term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies or byde novo synthesis using recombinant DNA methodologies such as singlechain Fv.

Antibodies are commonly referred to according their targets. While thenomenclature varies, one of skill in the art will be familiar andunderstand that several names can be applied to the same antibody. Forexample, an antibody specific for IgM can be called “anti-IgM,” “IgMantibody,” “anti-IgM antibody,” etc.

The terms “antigen,” “immunogen,” “antibody target,” “target analyte,”and like terms are used herein to refer to a molecule, compound, orcomplex that is recognized by an antibody, i.e., can be specificallybound by the antibody. The term can refer to any molecule that can bespecifically recognized by an antibody, e.g., a polypeptide,polynucleotide, carbohydrate, lipid, chemical moiety, or combinationsthereof (e.g., phosphorylated or glycosylated polypeptides, etc.). Oneof skill will understand that the term does not indicate that themolecule is immunogenic in every context, but simply indicates that itcan be targeted by an antibody.

Antibodies bind to an “epitope” on an antigen. The epitope is thelocalized site on the antigen that is recognized and bound by theantibody. Epitopes can include a few amino acids or portions of a fewamino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, orportions of those amino acids. In some cases, the epitope includesnon-protein components, e.g., from a carbohydrate, nucleic acid, orlipid. In some cases, the epitope is a three-dimensional moiety. Thus,for example, where the target is a protein, the epitope can be comprisedof consecutive amino acids, or amino acids from different parts of theprotein that are brought into proximity by protein folding (e.g., adiscontinuous epitope). The same is true for other types of targetmolecules that form three-dimensional structures. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996).

The terms “specific for,” “specifically binds,” and like terms refer toa molecule (e.g., antibody or antibody fragment) that binds to itstarget with at least 2-fold greater affinity than non-target compounds,e.g., at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 20-fold, 25-fold, 50-fold, or 100-fold greater affinity. Forexample, an antibody that specifically binds a given antibody targetwill typically bind the antibody target with at least a 2-fold greateraffinity than a non-antibody target. Specificity can be determined usingstandard methods, e.g., solid-phase ELISA immunoassays (see, e.g.,Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity).

The term “binds” with respect to an antibody target (e.g., antigen,analyte), typically indicates that an antibody binds a majority of theantibody targets in a pure population (assuming appropriate molarratios). For example, an antibody that binds a given antibody targettypically binds to at least ⅔ of the antibody targets in a solution(e.g., at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100%). One of skill will recognize that some variability willarise depending on the method and/or threshold of determining binding.

The terms “label,” “detectable label, “detectable moiety,” and liketerms refer to a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, chemical, or other physical means. Forexample, useful labels include fluorescent dyes (fluorophores),luminescent agents, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, ³²P and other isotopes, haptens,and proteins which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The term includes combinations of singlelabeling agents, e.g., a combination of fluorophores that provides aunique detectable signature, e.g., at a particular wavelength orcombination of wavelengths. Any method known in the art for conjugatinglabel to a desired agent may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

The term “positive,” when referring to a result or signal, indicates thepresence of an analyte or item that is being detected in a sample, inthis case, antibody indicative of syphilis infection. The term“negative,” when referring to a result or signal, indicates the absenceof an analyte or item that is being detected in a sample. Positive andnegative are typically determined by comparison to at least one control,e.g., a threshold level that is required for a sample to be determinedpositive, or a negative control (e.g., a known blank).

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters, and willunderstand which controls are valuable in a given situation and be ableto analyze data based on comparisons to control values. Controls arealso valuable for determining the significance of data. For example, ifvalues for a given parameter are variable in controls, variation in testsamples will not be considered as significant.

A “calibration control” is similar to a positive control, in that itincludes a known amount of a known analyte. In the case of a multiplexassay, the calibration control can be designed to include known amountsof multiple known analytes. The amount of analyte(s) in the calibrationcontrol can be set at a minimum cut-off amount, e.g., so that a higheramount will be considered “positive” for the analyte(s), while a loweramount will be considered “negative” for the analyte(s). In some cases,multilevel calibration controls can be used, so that a range of analyteamounts can be more accurately determined. For example, an assay caninclude calibration controls at known low and high amounts, or knownminimal, intermediate, and maximal amounts.

Controls can also be designed to ensure integrity of the sample (e.g.,to detect a component in a given sample type that is known to beuniversally present at a certain level), or the integrity of a signal(e.g., to detect a fluorophore added in a known amount). These controlscan be internal (run in the same sample that is being tested), orexternal (run separate from the sample that is being tested). In someembodiments, the presently disclosed assays include a control for samplequality. The sample quality control can include a solid support coatedwith a reagent that binds a blood component such as a clotting factor,albumin, globulin, or fibrinogen (e.g., anti-Factor XIII, see, e.g.,WO2013192445). This component (e.g., a serum verification bead or SVB)ensures that serum or plasma is present and of a baseline quality. Thesample quality control can also include a reagent blank bead (RBB)alone, or in combination with the solid support coated with a reagentthat binds a blood component. This component serves to identify and/orquantify non-specific binding in the sample. In some embodiments, thepresently disclosed assays include a signal quality control, e.g., aloneor in combination with the sample quality control. The signal qualitycontrol can be a solid support with an intrinsic fluorescence, or coatedwith a known amount of fluorophore. This component (e.g., internalstandard bead or ISB) can be useful for detecting and compensating fordetector fluctuations that may occur during analysis.

The term “diagnosis” refers to a relative probability that a subject hasan infection, disorder or disease. Similarly, the term “prognosis”refers to a relative probability that a certain future outcome may occurin the subject. For example, in the context of the present disclosure,prognosis can refer to the likelihood that an individual will beinfected in the future (e.g., unlikely if immunized). The terms are notintended to be absolute, as will be appreciated by any one of skill inthe field of medical diagnostics.

“Subject,” “patient,” “individual” and like terms are usedinterchangeably and refer to, except where indicated, mammals such ashumans and non-human primates, as well as rabbits, rats, mice, goats,pigs, and other mammalian species. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision. Apatient can be an individual that is seeking treatment, monitoring,adjustment or modification of an existing therapeutic regimen, etc.

C. MULTIPLEX ASSAYS FOR DETECTING TREPONEMAL AND NONTREPONEMALANTIBODIES

The presently described assays involve detection of more than oneanalyte in a single assay, and are thus described as multiplex assays.The presently described assays include components for immobilizingmultiple analytes on distinguishable solid supports so that each of themultiple analytes can be identified and quantified by flow cytometry.Assay components and considerations include the solid supports and howto distinguish the different types of solid supports from one another(e.g., labels or other differentiation parameters), components tospecifically immobilize the desired analytes and remove other samplematerials, and labels for detecting and quantifying the desiredanalytes.

In this case, the multiplex assay includes a hydophobic component, e.g.,a solid support coupled to lipid antigen (e.g.,cardiolipin-lecithin-cholesterol), and a hydrophilic component e.g.,solid support coupled to a syphilis antigen (e.g., r15, r17, r47, ormixtures or fusions thereof). Beads are commonly functionalized forattachment to proteins (explained in more detail below), but attachmentof the lipid antigen in such a manner as to allow simultaneousprocessing with the syphilis antigen-coated beads is carried out withspecific considerations.

1 Coupling of Nontreponemal Antigens to Solid Supports

Nontreponemal Antigen Mixture (RPR or VDRL)

The nontreponemal antigen mixture can be in an ethanol solution ofcardiolipin (a diphosphatidylglycerol purified from beef heart orsynthetic), lecithin (from hen egg yolk, soybean, or synthetic), andcholesterol, wherein the weight ratio of cardiolipin, lecithin andcholesterol is in the range of 0.03-1 mg/mL, 0.01-3 mg/mL, and 0.1-10mg/mL respectively. Other organic solvents like methanol, chloroform,benzene and acetone could also be used to solubilize the nontreponemal(RPR or VDRL) antigen. The examples show use of VDRL antigen with 0.3mg/mL cardiolipin, 2.1 mg/mL lecithin, and 9 mg/mL cholesterol.

Nontreponemal Antigen/Bead Coupling

To ensure enough positive charges on the bead surface for ionicinteraction with the nontreponemal antigen mixture, the pH of thecoupling buffer can be below the pI of amine functionalized beads,usually between 5 to 8. Examples include MES buffer pH 6.1, phosphatebuffer pH 7.0, and MOPS buffer pH 7.4.

The positively charged ligand coupled to the bead can be an aminefunctional molecule, including but not limited to ethylenediamine,N,N-Diethylethylenediamine, other amine compounds, or polycationicpolymers such as but not limiting polyethyleneimine (PEI). PEI caninclude linear or branched polyethyleneimines that contain primary,secondary or tertiary amino groups with molecular weights ranging from1000 to 1,000,000 daltons, or polylysine and other copolymers withmolecular weights ranging from 1,000 to 300,000 daltons. In someembodiments, the polylysine is between 15,000 and 30,000 daltons.

2. Coupling of Treponemal Antigens to Solid Supports

The treponemal antigen (e.g., r15, r17, and r47, or subcombinations,mixtures, or fusions thereof) can be coupled to a solid support viacovalent bonding. In some embodiments, the solid support iscarboxylated, to allow for convenient addition of functional groups(e.g., carboxylated beads). In some embodiments, the carboxylatedsupport is activated and esterified before adding the treponemalantigen. Carboxyl activation can be achieved using a water solublecarbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMC), ordicyclohexylcarbodiimide (DCC). Esterification can be achieved, e.g.,using NHS, NHSS or HOBt.

After the carboxyl activation and esterification, the treponemal antigencan added to the actived surface in buffers with pH between 6-10.Examples include MES buffer pH 6.1, phosphate buffer pH 7.0, MOPS bufferpH 7.4, and carbonate buffer pH 9.0.

After the coupling, the solid support (e.g., beads) can be blocked inbuffers containing protein blockers such as BSA, casein, milk powder,mouse IgG, bovine gamma globulin (BGG), animal serum (goat, horse,murine). For example, the protein blocker(s) can be present in an amountranging from 0.1-10 weight/volume percent.

3. Storage Buffer

Beads coated with nontreponemal antigen can be suspended in an aqueousbuffer pH 5-10, e.g., 6-8. The nontreponemal antigen-coated beads can bestored with, or separate from beads coated with treponemal antigen.Various buffers such as Trizma, glycine, and phosphate can be used, withthe latter being most common. The concentration of buffer will generallybe in the range of approximately 0.001 to 0.1 M, e.g., 0.01-0.05 M. Thebuffer can contain a salt such as sodium chloride, e.g., in the range ofapproximately 0.01 to 0.5 M or 0.05-0.2 M. In some embodiments, thestorage buffer further comprises cholesterol.

Non-ionic detergents such as Tween-20 can exert a deleterious effect onthe hydrophobic micro environment on the nontreponemal antigen (e.g.,RPR) coated beads. This can lead to nontreponemal antigen instabilityand poor assay performance (e.g., reduced affinity of RPR-specificantibodies for antigen).

Accordingly, for storage or running the assay, non-ionic detergent canbe omitted and replaced with additives that minimize or mitigateinstability, and that stabilize the individual antigen(s) or improve theassay performance. Particularly, ethylenediaminetetraacetic acid (EDTA)can be employed in amounts of approximately 0.1 to 1.0 weight/volume(w/v) percent. CHAPS or other zwitterionic detergents such as CHAPSO,IGEPAL and Zwittergent 3-8, 3-10, 3-12, 3-14 and 3-16 can be employed,for example, in amounts of approximately 0.01 to 0.5 w/v percent. BSAcan be employed, for example, in amounts of approximately 0.01 to 3 w/vpercent. Glycerol can be employed, for example, in amounts ofapproximately 0.05 to 25 w/v percent. Sodium azide can be employed inamounts of approximately 0.005 to 0.1 w/v percent.

The conjugate reagent buffer optionally also include cholesterol inconcentrations of 30-150 μg/mL, 30-100 μg/mL, 30 μg/mL, 35 μg/mL, 40μg/mL or 50 μg/mL. In some embodiments, the cholesterol is bovinecholesterol.

4. Assay Buffers

Assay buffers (e.g., for carrying out antibody-binding assays with thecombined beads) can include a buffer such as phosphate, MOPS,triethanolamine (TEA), HEPES, TES, EPPS or Trizma. In some embodiments,the buffer is in a concentration of 10-100 mM, for example, at pH 6.0 to8.0. The buffer can further comprise sodium chloride, e.g., at 50-300mM. In some embodiments, the buffer contains protein stabilizers such asBSA, casein, milk powder, mouse IgG, bovine gamma globulin (BGG), animalserum (goat, horse, murine). In some embodiments, the proteinstabilizer(s) is in an amount ranging from 0-10% of the final volume.Buffers may contain preservatives including but not limited to ProClin300, ProClin 900, sodium benzoate and sodium azide.

All buffers can contain ionic and zwitterionic detergents such as CHAPS,IGEPAL, CHAPSO and Zwittergent 3-8, 3-10, 3-12, 3-14 and 3-16, e.g., inconcentrations between 0.05 to 1.0 or 0.1-0.5 volume percent.

5. Wash Buffers

Wash buffers (e.g., for removing non-reactive reagent and non-specificbindings on beads after each incubation step) can include a buffer suchas phosphate, MOPS, triethanolamine (TEA), HEPES, TES, EPPS or Trizma.The buffer can be at a concentration of, e.g., 10-100 mM, e.g., at pH6.0 to 8.0. The buffer can further comprise sodium chloride, e.g., at50-300 mM. In some embodiments, the wash buffer may contain Tween-20 ornon-ionic detergent such as Tween 40, Tween 60, and Triton X-100, inconcentrations between 0.05 to 1.0 or 0.1-0.5 volume percent.

6. Controls

Controls (e.g., internal controls) can be used with the presentlydescribed assays. An internal standard (e.g., internal standard bead orISB) can be used to rectify fluctuations in signals due to voltagespikes or progressive changes in detector temperature or other factorsthat affect detector performance. The internal standard has inherentfluorescence that is independent of assay chemistry. For example, theinternal standard can be derivatized with tetramethylrhodamine, or otherstable label that does not interfere with or overlap with labels beingused to detect sample components.

A sample control can also be used to ensure that sample, e.g., serum orplasma, has been introduced in the reaction and has not been diluted. Asample control can include a solid support coated with an agent thatbinds to a component of the sample, for example, a serum verificationbead (SVB). The serum verification support can be coated withanti-Factor XIII antibody. Soluble Factor XIII (β subunit) can bemeasured in a sandwich-type immunoassay. A sample control can alsoinclude a reagent blank control, e.g., a solid support that is notcoated with anything, to detect background binding of sample to thesolid support.

7. Solid Supports

As explained above, the presently described multiplex assays involve useof a solid support, typically particles (also referred to asmicroparticles or beads). For detection by flow cytometry, particlesthat emit autofluorescence should be avoided since this will increasebackground signal and render them unsuitable. Particles created bystandard emulsion polymerization from a variety of starting monomersgenerally exhibit low autofluorescence, while those that have beenmodified to increase porosity (“macroporous” particles) exhibit highautofluorescence. Autofluorescence in such particles further increaseswith increasing size and increasing percentage of divinylbenzenemonomer.

The size range of the microparticles can vary and particular size rangesare not critical. In most cases, the aggregated size range of themicroparticles lies within the range of from about 0.3 micrometers toabout 100 micrometers in particle diameter, e.g., within the range offrom about 0.5 micrometers to about 40 micrometers.

Magnetic particles are commonly used in the art, and can make separationand wash steps more convenient for the presently described assays.“Magnetic particles,” “magnetically responsive material,” “magneticbeads,” and like terms denote a material that responds to a magneticfield. Magnetically responsive materials include paramagnetic materials(e.g., iron, nickel, and cobalt, as well as metal oxides such as Fe₃O₄,BaFe₁₂O₁₉, CoO, NiO, Mn₂O₃, Cr₂O₃, and CoMnP), ferromagnetic materials,ferrimagnetic materials, and metamagnetic materials. Rather thanconstituting the entire microparticle, the magnetically responsivematerial typically constitutes one component of the microparticle, whilethe remainder consists of a polymeric material which can be chemicallyderivatized to permit attachment of an assay reagent (e.g., antigen orantibody). Attachment of the assay reagent can be either direct (e.g.,covalent) or indirect (e.g., through ionic or other affinityinteractions).

Methods of, and instrumentation for, applying and removing a magneticfield as part of an assay are known to those skilled in the art andreported in the literature. Examples of literature reports are Forrestet al., U.S. Pat. No. 4,141,687; Ithakissios, U.S. Pat. No. 4,115,534;Vlieger et al., Analytical Biochemistry 205:1-7 (1992); Dudley, Journalof Clinical Immunoassay 14:77-82 (1991); and Smart, Journal of ClinicalImmunoassay 15:246-251 (1992).

The polymeric matrix that forms the microparticle can be any materialthat is compatible with the presently described assays. The matrixshould be inert to the components of the biological sample and to theassay reagents, have minimal autofluorescence, be solid and insoluble inthe sample and in any other reagents or washes used in the assay, andcapable of affixing an assay reagent to the microparticle. Examples ofsuitable polymers are polyesters, polyethers, polyolefins, polyalkyleneoxides, polyamides, polyurethanes, polysaccharides, celluloses, andpolyisoprenes. Crosslinking is useful in many polymers for impartingstructural integrity and rigidity to the microparticle.

Functional groups for attachment of an assay reagent (e.g., antigen orantibody) can be incorporated into the polymer structure by conventionalmeans. Examples of suitable functional groups are amine groups, ammoniumgroups, hydroxyl groups, carboxylic acid groups, and isocyanate groups.Assay reagent is typically covalently bound to the solid phase surface,either directly or indirectly, e.g., with a linking group. Linkinggroups can be used as a means of increasing the density of reactivegroups on the solid phase surface and decreasing steric hindrance toincrease the range and sensitivity of the assay, or as a means of addingspecific types of reactive groups to the solid phase surface to broadenthe range of types of assay reagents that can be affixed to the solidphase. Examples of suitable useful linking groups are polylysine,polyaspartic acid, polyglutamic acid and polyarginine.

Microparticles of different types in a multiplex assay can bedistinguished from one another, e.g., by size, weight, light scatter orabsorbance, reflectance, shape, or label, e.g., fluorescent label.

Where microparticle size is used as a differentiation factor(distinguishing characteristic), the widths of the size subranges andthe spacing between mean diameters of adjacent subranges are selected topermit differentiation of different types of microparticles by flowcytometry, as will be apparent to those skilled in the use of andinstrumentation for flow cytometry. Typically, a subrange for a givenmean diameter is about ±5% CV or less of the mean diameter, where CV isthe coefficient of variation and is defined as the standard deviation ofthe particle diameter divided by the mean particle diameter times 100percent. The mean diameters of subranges for different types ofparticles are generally spaced apart by at least about 6% of the meandiameter of one of the subranges, e.g., at least about 8% or 10% of themean diameter of one of the subranges.

Light scatter can also be used to distinguish different types ofmicroparticles. Side angle light scatter varies with particle size,granularity, absorbance and surface roughness, while forward angle lightscatter is mainly affected by size and refractive index. Varying any ofthese qualities can result in light scatter differences that can serveas a means of distinguishing the various groups.

Still another example of a differentiation parameter is absorbance. Whenlight is applied to particles, the absorbance of the light by theparticles is indicated mostly by a change in the strength of thelaterally (side-angle) scattered light while the strength of theforward-scattered light is relatively unaffected. Consequently, thedifference in absorbance between various colored dyes associated withthe particles is determined by observing differences in the strength ofthe laterally scattered light.

A wide array of parameters or characteristics can be used asdifferentiation parameters to distinguish the particles of one groupfrom those of another. The differentiation parameters may arise fromparticle size, composition, physical characteristics that affect lightscattering, excitable fluorescent dyes or colored dyes that impartdifferent emission spectra and/or scattering characteristics to theparticles, or from different concentrations of one or more fluorescentdyes.

When the distinguishable characteristic is a fluorescent dye or color,it can be coated on the surface of the microparticle, embedded in themicroparticle, or bound to the molecules of the microparticle material.Thus, fluorescent microparticles can be manufactured by combining thepolymer material with the fluorescent dye, or by impregnating themicroparticle with the dye. Microparticles with dyes alreadyincorporated and thereby suitable for use in the present invention arecommercially available, from suppliers such as Spherotech, Inc.(Libertyville, Ill., USA) and Molecular Probes, Inc. (Eugene, Oreg.,USA). A list of vendors of flow cytometric products can be found, e.g.,at the website of molbio.princeton.edu/facs/FCMsites.html.

8. Detectable Labels

The presently described assays can have several labeled components,e.g., beads, secondary antibodies (e.g., labeled, anti-Ig antibodies),signal quality and sample quality controls. Labels can be any substanceor component that directly or indirectly emits or generates a detectablesignal. In some embodiments, the labels are fluorophores, many of whichare reported in the literature and thus known to those skilled in theart, and many of which are readily commercially available. Literaturesources for fluorophores include Cardullo et al., Proc. Natl. Acad. Sci.USA 85: 8790-8794 (1988); Dexter, J. of Chemical Physics 21: 836-850(1953); Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992);Selvin, Methods in Enzymology 246: 300-334 (1995); Steinberg, Ann. Rev.Biochem., 40: 83-114 (1971); Stryer, Ann. Rev. Biochem. 47: 819-846(1978); Wang et al., Tetrahedron Letters 31: 6493-6496 (1990); and Wanget al., Anal. Chem. 67: 1197-1203 (1995).

The following are examples of fluorophores that can be used as labels:

-   -   4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid        acridine    -   acridine isothiocyanate    -   5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)    -   4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate    -   N-(4-anilino-1-naphthyl)maleimide    -   anthranilamide    -   BODIPY    -   Brilliant Yellow    -   coumarin    -   7-amino-4-methylcoumarin (AMC, Coumarin 120)    -   7-amino-4-trifluoromethylcoumarin (Coumaran 151)    -   cyanine dyes    -   cyanosine    -   4′,6-diaminidino-2-phenylindole (DAPI)    -   5′, 5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)    -   7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin        diethylenetriamine pentaacetate    -   4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid    -   4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid    -   5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,        dansylchloride)    -   4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)    -   4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC)    -   eosin    -   eosin isothiocyanate    -   erythrosin B    -   erythrosin isothiocyanate    -   ethidium    -   5-carboxyfluorescein (FAM)    -   5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)    -   2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)    -   fluorescein    -   fluorescein isothiocyanate    -   fluorescamine    -   IR144    -   IR1446    -   Malachite Green isothiocyanate    -   4-methylumbelliferone    -   ortho cresolphthalein    -   nitrotyrosine    -   pararosaniline    -   Phenol Red    -   phycoerythrin (including but not limited to B and R types)    -   o-phthaldialdehyde    -   pyrene    -   pyrene butyrate    -   succinimidyl 1-pyrene butyrate    -   quantum dots    -   Reactive Red 4 (Cibacron™ Brilliant Red 3B-A)    -   6-carboxy-X-rhodamine (ROX)    -   6-carboxyrhodamine (R6G)    -   lissamine rhodamine B sulfonyl chloride rhodamine    -   rhodamine B    -   rhodamine 123    -   rhodamine X isothiocyanate    -   sulforhodamine B    -   sulforhodamine 101    -   sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)    -   N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)    -   tetramethyl rhodamine    -   tetramethyl rhodamine isothiocyanate (TRITC)    -   riboflavin    -   rosolic acid    -   lanthanide chelate derivatives

A prominent group of fluorophores for immunoassays are fluorescein,fluorescein isothiocyanate, phycoerythrin, rhodamine B, and Texas Red(sulfonyl chloride derivative of sulforhodamine 101). Any of thefluorophores in the list preceding this paragraph can be used in thepresently described assays, either to label the microparticle, or tolabel a binding agent (e.g., an antibody or streptavidin). Fluorochromescan be attached by conventional covalent bonding, using appropriatefunctional groups on the fluorophores and on the microparticle orbinding agent. The recognition of such groups and the reactions to formthe linkages will be readily apparent to those skilled in the art. Otherlabels that can be used in place of the fluorophores are radioactivelabels and enzyme labels. These are likewise known in the art.

D. DETECTION OF TREPONEMAL AND NONTREPONEMAL ANTIBODIES

Once solid supports are coated with treponemal and nontreponemalantigens as described above, the coated solid supports are contactedwith sample, e.g., biological sample from an individual suspected ofhaving been exposed to syphilis, e.g., in the presence of assay bufferas described above. Unbound sample can be washed away. In this assay, 3,4 or 5 wash steps can be used. Labeled anti-Ig antibodies are added toallow for detection of antibodies from the sample bound to the solidsupports. Where the sample is from a human, labeled anti-human Igantibodies are used. For example, a mixture of anti-IgG and anti-IgMantibodies, or an antibody that binds both IgG and IgM, can be used.

Flow cytometry can be used to detect size, weight, light scatter, andfluorescence of assay components. Flow cytometry methods andinstrumentation are known in the art. Descriptions of instrumentationand methods can be found, e.g., in Introduction to Flow Cytometry: ALearning Guide (2000) Becton, Dickinson, and Company; McHugh, “FlowMicrosphere Immunoassay for the Quantitative and Simultaneous Detectionof Multiple Soluble Analytes,” Methods in Cell Biology 42, Part B(Academic Press, 1994).

Flow cytometry in general involves passage of a suspension (e.g., beadsor microparticles) as a stream past a light beam and electro-opticalsensors, in such a manner that only one particle at a time passesthrough the region. As each particle passes this region, the light beamis perturbed by the presence of the particle, and the resultingscattered and fluorescent light is detected. The optical signals areused by the instrumentation to identify the subgroup to which eachparticle belongs, along with the presence and amount of label, so thatindividual assay results are achieved. Descriptions of instrumentationand methods for flow cytometry are found in the literature. Examples areMcHugh, “Flow Microsphere Immunoassay for the Quantitative andSimultaneous Detection of Multiple Soluble Analytes,” Methods in CellBiology 42, Part B (Academic Press, 1994); McHugh et al.,

“Microsphere-Based Fluorescence Immunoassays Using Flow CytometryInstrumentation,” Clinical Flow Cytometry, Bauer, K. D., et al., eds.(Baltimore, Md., USA: Williams and Williams, 1993), pp. 535-544; Lindmoet al., “Immunometric Assay Using Mixtures of Two Particle Types ofDifferent Affinity,” J. Immunol. Meth. 126: 183-189 (1990); McHugh,“Flow Cytometry and the Application of Microsphere-Based FluorescenceImmunoassays,” Immunochemica 5: 116 (1991); Horan et al., “Fluid PhaseParticle Fluorescence Analysis: Rheumatoid Factor Specificity Evaluatedby Laser Flow Cytophotometry,” Immunoassays in the Clinical Laboratory,185-189 (Liss 1979); Wilson et al., “A New Microsphere-BasedImmunofluorescence Assay Using Flow Cytometry,” J. Immunol. Meth. 107:225-230 (1988).

Signal can also be detected by a fluorescent imager (e.g., a LuminexMagpix®), e.g., for assays run in an ELISA format (e.g., on a microtiterplate). In addition, for beads or plates, signal detection can becarried out using an addressable array (e.g., an Illumina detector orsimilar system).

E. KITS

Also provided are kits for detecting treponemal and nontreponemalantibodies as described herein. In some embodiments, the kit comprises(i) solid supports (e.g., beads) coupled to nontreponemal antigen and(ii) solid supports (e.g., beads) coupled to treponemal antigen. In someembodiments, the solid supports of (i) and (ii) are in the same storagecompartment (e.g., tube or vial). In some embodiments, the solidsupports of (i) and (ii) are in the separate storage compartments. Insome embodiments, the solid supports of (i) are PEI-functionalized.

The kit can also comprise other reagents, e.g., secondary antibodies,assay buffers, wash buffers, and stock solutions (e.g., concentratedbuffers). In some embodiments, the secondary antibody is labeledanti-human Ig as described herein. In some embodiments, the kit caninclude consumables such as multiwell plates or tubes for carrying outthe assay.

F. EXAMPLES Example 1

This example illustrates the performance of the combination treponemaland nontreponemal test (detecting antibodies against syphilis antigenand lipoidal antigen, respectively) against comparators on a set of 113clinical serum samples with known disease stages and treatment status,and 285 syphilis test ordered samples. Patient samples were tested forthe presence of treponemal and nontreponemal antibodies using theBioPlexi™ 2200 platform as shown in FIG. 1.

As shown in FIG. 1, beads coated with a recombinant treponemal r17-r47fusion protein (Rec Tp17/r47 fusion) and PEI-functionalized beads coatedwith non-treponemal antigens cardiolipin, lecithin, and cholesterol weremixed in assay buffer (50 mM MOPS, 150 mM NaCl, 0.05% BSA, 10 mM EDTA,0.1% CHAPS, 20% glycerol, and 0.095% sodium azide). The two sets ofanalyte beads are distinguishable via different labels (R17 vs. R21 fortreponemal vs. nontreponemal beads). Two sets of control beads wereused, a serum verification bead coated with FXIII and an internalstandard bead derivatized with tetramethylrhodamine. These aredistinguishable due to a unique combination of fluorescent dyes(classification dyes). Samples were diluted with a sample dilutionbuffer (50 mM TEA, 150 mM NaCl, 75 mM MgCl₂, 3% BSA, 0.03% mouse IgG,0.47% gamma globulin, 0.0015% PSMA, 10 IU/L aprotinin, 0.1% sodiumbenzoate, 0.095% sodium azide, 0.3% ProClin, at pH 7.4). Diluted samplewas added, each sample potentially including anti-treponemal antibodies(IgG anti-Syph Ab and IgM anti-Syph Ab) and anti-nontreponemalantibodies (IgG anti-CrL Ab and IgM anti-CrL Ab). After 20 minutesincubation, the beads were washed in wash buffer containing 50 mM PBSand 0.1% Tween-20. The beads where then incubated with a conjugatereagent (labeled secondary antibody specific for human IgG and IgM). Theconjugate reagent in this case was a bispecific anti-human IgG andanti-human IgM labeled with PE in a solution of 50 mM sodium phosphate,150 mM NaCl, 1% BSA, 0.1% mouse IgG, 3.3 IU/L aprotinin, 0.1% sodiumbenzoate, 0.095% sodium azide, and 0.3% ProClin at pH 7.4. After 10minutes incubation, the beads were washed a second time in wash bufferincluding 50 mM PBS and 0.1% Tween-20.

Subsequently, washed beads were suspended in sheath fluid (phosphatebuffer saline (PBS) containing 0.3% ProClin 300 and 0.095% sodium azide)and sent to the detector. The fluorescent dye signature and the presenceof bound antibodies were detected for each bead by flow cytometer.Treponemal and/or nontreponemal specific signal associated with eachbead was compared to matched controls and results reported. Dividing thetest bead signal by the ISB signal (fluorescent ratio) can rectifysignal within and between runs including controls for detector stabilityand fluctuations.

A comparison of results obtained from combo test and those fromcomparators are listed in Tables 1 and 2.

TABLE 1 BioPlex syphilis combo test Comparator test Syphilis TreatmentNumer of Treponemal assay Non-treponemal assay Treponemal assayNon-treponemal assay category Status Samples R* NR** R NR R NR R NRPrimary Untreated 8 8 0 7 1 8 0 7 1 Treated 24 21 3 20 4 21 3 17 7Secondary Untreated 17 17 0 14 3 17 0 17 0 Treated 25 25 0 16 9 25 0 1510 Latent Untreated 7 7 0 5 2 7 0 6 1 Treated 32 31 1 21 11 31 1 21 11Total Medically diagnosed 113 109 4 83 30 109 4 83 30 syphilis samplesSyphilis negative samples 285 1 284 3 282 1 284 0 285 Note: *R isreactive, **NR is non-reactive.

TABLE 2 BioPlex syphilis combo test Treponemal assay Non-treponemalassay Numer of % Positive % Negative % Positive % Negative Syphiliscategory Samples Agreement Agreement Agreement Agreement Medicallydiagnosed 113 100% N/A 92.7% N/A syphilis samples (109/109) (77/83)Syphilis test ordered 285 N/A 99.6% N/A 98.9% samples (283/284)(282/285)

For medically diagnosed syphilis samples, excellent agreement wasobserved between the clinically diagnosed untreated and treated primarysecondary and latent infections and the combination treponemal test aswell as the comparator test. The combination test detectednon-treponemal antibody in 20 of 24 treated primary disease specimens,14 of 17 treated secondary disease specimens, 16 of 25 untreatedsecondary disease specimens, and 5 of 7 untreated latent diseasesamples; while the comparator non-treponemal test detected in 17 of 24treated primary disease, 17 of 17 treated secondary disease, 15 of 25untreated secondary disease, and 6 of 7 untreated latent diseasespecimens. Of the 285 Syphilis negative samples, 284 tested negative fortreponemal antibodies and 282 for non-treponemal antibodies. Overall,the combination test exhibits 100% sensitivity and 99.6% specificity fortreponemal antibody assay, and 92.7% sensitivity and 98.9% specificityfor non-treponemal antibody assays respectively.

EXAMPLE 2

In a second example the performance of the combination treponemal andnon-treponemal test detecting antibodies (both IgG and IgM for bothtreponemal and non-treponemal) against the treponemal and lipoidalantigen was evaluated using 228 RPR/VDRL positive serum samples, 226Syphilis test ordered serum samples and 122 clinical serum samples withknown disease stages and treatment status. Subsequently, the resultsfrom the combination test were compared head-to-head with the DiasorinLiaison™ treponema assay or Fujirebio Serodia™ TPPA assay and BDMacro-Vue™ RPR card test (Tables 3-5).

For the RPR/VDRL positive sample cohort, a positive agreement of 99.5%(211/212) and a negative agreement of 100% (14/14) was observed betweenthe treponemal component of the combination test and the Liaison™Treponema assay, Table 3a. In contrast, a comparison between thenon-treponemal component of the combination test and BD Macro-Vue RPRcard test revealed a positive agreement of 99.1% (217/219) and anegative agreement of 71.4% (5/7), Table 3b.

Table 3. Head-to-head comparison between the combination assaytreponemal component and Liaison™ Treponema assays (3a) and between thecombination non-treponemal assay component and BD Macro-vue™ RPR assays(3b) using 226 RPR/VDRL Positive sample cohort.

TABLE 3a Liaison ™ Treponema Positive Negative Equivocal TotalCombination Positive 211 0 0 211 Assay Negative 1 14 0 15 TreponemalEquivocal 0 0 0 0 Total 212 14 0 226

TABLE 3b BD Macro-Vue ™ RPR Positive Negative Total Combination AssayPositive 217 2 219 Non-treponemal Negative 2 5 7 Total 219 7 226

Under similar assay conditions the test ordered sample cohort displayeda positive agreement of 93.3% (28/32) and a negative agreement of 97.5%(193/198) between the treponemal component of the combiantion assay andthe Liaison™ Treponema assay, Table 4a. Within the same population thepositive and negative agreement between the non-treponemal component ofthe combination assay and manual BD Macro-Vue RPR card test was 95%(19/20) and 99.5% (207/208) respectively, Table 4b.

Table 4. Head-to-head comparison between the combination assaytreponemal component and Liaison™ Treponema assay (2a) and between thecombination non-treponemal assay component and BD Macro-vue RPR assays(2b) using 228 test ordered sample cohort.

TABLE 4a Liaison ™ Treponema Positive Negative Equivocal TotalCombination Assay Positive 28 4 0 32 Treponemal Negative 0 193 1 194Equivocal 1 1 0 2 Total 29 198 1 228

TABLE 4b BD Macro-Vue ™ RPR Positive Negative Total Combination AssayPositive 19 1 20 Non-treponemal Negative 1 207 208 Total 219 7 226

In order to assess the clinical sensitivity of the combination assay, atotal of 122 samples with known clinical status were tested, Table 5.Samples were from treated and untreated patients with primary, secondaryand latent infection. While untreated samples exhibit an overallsensitivity of 97.6% (40/41), treated samples revealed a sensitivity of95.1% (77/81) using the combination treponemal assay. When the samesample cohort was tested in parallel with the Fujirebio Serodia™ TPPAassay, an exactly equivalent sensitivity was observed for untreated andtreated groups. On the other hand, the combination non-treponemal assayrevealed an overall sensitivity of 95.1% (39/41) for the untreated groupcompared to 86.4% (70/81) for the treated group. These results are incontrast to the BD Macro-Vue™ card test that demonstrated an overallsensitivity of 95.1% for the untreated group and 75.3% for the treatedgroup. Collectively, these results suggest higher sensitivity of thecombination non-treponemal assay.

TABLE 5 Comparison of the combination assay with the Fujirebio Serodia ™TPPA and BD Macro-Vue ™ RPR card assays using 122 clinicallycharacterized primary, secondary and latent syphilis samples. TreponemalNon-treponemal Combination Combination Assay Assay BD Micro- GroupTreponemal Serodia ™ TPPA Non-treponemal Vue ™ RPR Untreated 97.6%(40/41) 97.6% (40/41) 95.1% (39/41) 95.1% (39/41) Treated 95.1% (77/81)95.1% (77/81) 86.4% (70/81) 75.3% (61/81)

EXAMPLE 3 Functionalization of Bead Surface for Coating withNontreponemal Antigen

FIG. 2 shows an example of functionalizing a bead with amine ligand toallow for ionic interaction with lipid RPR antigen mixture. Positivelycharged ligand is first bound to the carboxyl beads via either passiveadsorption or covalent binding to introduce positive charges on the beadsurface.

Lipids (with net negative charge due to the presence of phosphate group)could not be coupled directly on activated carboxylated beads. In orderto bind cardiolipin, cholesterol, and lecithin (a trio of lipids) wechanged the net charge on magnetic beads by covalently coupling apositively charged group such as polyethylenimine (PEI). Beads with thecationic imino group were then incubated with negatively charged lipids.The interaction produces a stable antigen coating the bead surface asevaluated by stability studies.

As explained above, the positively charged ligand can be an aminefunctional molecule. We tested the ability of nontreponemal antigen(RPR) to bind to beads coated with ethylenediamine, polyethyleneimine(PEI), and poly-lysine.

TABLE 6 Performance of beads functionalized with indicated componentPoly- Sample ID Ethylenediamine Poly(ethyleneimine) Lysine RPR SS0297856175 514 546 RPR SS0297418 289 866 902 positive JV0144150 161 228 254(Titer JK0490505 116 576 470 64) JK0489744 114 502 498 HB70942 72 62 101Neg- HB70946 94 62 110 ative HB70953 53 49 67 HB70958 51 57 75 HB7096455 57 92

Beads were functionalized with ethylenediamine, poly(ethyleneimine), orpoly-lysine and coated with RPR. The coated beads were then exposed toRPR antibody positive samples (top 5 rows) or antibody negative samples(bottom 5 rows). In this assay, poly(ethyleneimine) (PEI) functionalizedbeads generally performed the best, with higher reading for positivesamples and low background readings for negative samples.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, interne sources, patents,patent applications, and accession numbers cited herein are herebyincorporated by reference in their entireties for all purposes.

What is claimed is:
 1. A kit comprising: (i) beads coated withcardiolipin-lecithin-cholesterol antigen; and (ii) beads coated withTreponema pallidum antigen.
 2. A method of detecting the presence ofsyphilis antibodies in a sample comprising: (a) contacting the samplewith (i) beads coated with cardiolipin-lecitin-cholesterol antigen and(ii) beads coated with Treponema pallidum antigen in the samecompartment, thereby forming a sample-bead mixture; (b) washing thesample-bead mixture to remove unbound sample; (c) contacting thesample-bead mixture with a labeled, anti-human immunoglobulin (Ig)antibody; and (d) detecting the presence of syphilis antibodies bydetecting the label on the anti-human Ig antibody.
 3. The method ofclaim 2, further comprising washing to remove unbound labeled anti-humanIg antibody between steps (c) and (d).
 4. The method of claim 2, whereinthe contacting step (a) and/or (c) is carried out in the absence ofnon-ionic detergent; or the contacting step (a) and/or (c) is carriedout in the presence of ionic or zwitterionic detergent.
 5. The method ofclaim 2, wherein the labeled, anti-human Ig antibody is specific for IgGor IgM, optionally wherein the labeled, anti-human immunoglobulinantibody is an antibody conjugate that binds human IgG and human IgM. 6.The method of claim 2, wherein step (c) comprises contacting thesample-bead mixture with a first labeled, anti-human Ig antibodyspecific for IgG and a second labeled, anti-human Ig antibody specificfor IgM.
 7. The method of claim 6, wherein the first and secondanti-human Ig antibodies have different labels.
 8. The method of claim2, wherein the Treponema pallidum antigen is a fusion of r17 and r47;and/or the beads of (ii) are attached to the Treponema pallidum antigenvia a covalent interaction.
 9. The method of claim 2, wherein the beadsof (i) are cationic, and the cardiolipin-lecitin-cholesterol antigen isattached to the beads via an ionic interaction; and/or the beads of (i)are functionalized with polyethylenimine.